Single cell analysis is necessary to fully capture cell heterogeneity. This may be particularly true for the analysis of cancer cells. A change from two- to three-dimensional environments may radically affect cell behavior, such as gene expression and communication. Currently it is difficult to study the size and shape of freely suspended single cells, particularly Circulating Tumor Cells and disseminating Cancer Stem Cells.
The heterogeneity, i.e., non-uniformity, found in cancer cell populations, and the ubiquitous cell differentiation, has led to increased interest in individual cell studies. Historically, a tumor was thought to originate from the successive divisions of a single ‘mother cell’, leading to the assumption that all the cells in a tumor shared the same genetic code. However, recent findings have altered this theory, stressing the need for tools that can monitor and track single cells in a high throughput fashion. Currently, standard assays performed on cell populations make individual patterns difficult to access, due to effects of averaging. Flow cytometry, for instance, has been widely used in the last 20 years, for its ability to perform fast analysis on a very high number of cells at a time (e.g., 10,000 cells/s). Time point analysis can also be performed using this technique, but it is not possible to track each cell individually.
It is especially important that a minority of cells, such as stem cells, whose behavior could be considered to be statistically irrelevant compared to the large majority of the population, can have a critical biological and medical impact. For example, the use of the Imatinib drug that targets the BCR-abl fusion protein in patients with chronic myelogenous leukemia (CML) first seemed to be one of the most successful targeted therapies. However, the treatment does not eliminate the CML stem cells, and with the withdrawal of Imatinib the disease reappeared. As a consequence, the focus on cell-to-cell variations has also allowed important breakthroughs in understanding cell differentiation, drug response, protein mechanisms and dynamics, as well as the important role played by stem cells, especially for cancer stem cells. Metastasis relies on cancer cells circulating in the vascular network. The cells responsible for cancer propagation to secondary tumor sites are extremely rare (a few cells per million in the blood), and they go through a circulating stage before populating other tissues. Along with single cell analysis, three dimensional assays also permit a better comprehension of cellular dynamics, by narrowing the gap between in vitro and in vivo behavior. The previously mentioned single cell analysis techniques are all restricted by their confinement of the cell to two dimensions.
Magnetic microbeads have been used in a variety of methods as labels to indicate the presence of a biological molecule. Typically, these assays involve capturing the target of interest (e.g., an antigen or an antibody) on a surface, and using antibody-labeled magnetic beads or particles to bind to the target. The presence of the magnetic labels can be measured in a variety of ways, including changes in magnetoresistance, relaxation time, translational motion, and particle agglutination. Unfortunately, the sensitivity of these methods, as well as their flexibility for use with a variety of analytes, has been limited.
In response, a label-acquired magneto-rotation technique has been developed (referred to as magnetic-label-acquired rotation or MLAR), in which the target facilitates the binding of one or more magnetic labels (e.g., beads) to a nonmagnetic substrate (e.g., sphere) that is typically able to rotate (e.g., floating, suspended, etc.), and the rotational frequency of the resulting sandwich complex in a rotating magnetic field depends on the number of attached magnetic label beads. Label-acquired magnetorotation is derived from asynchronous magnetic bead rotation (AMBR), in which magnetic particles rotate at a different rate than that of a driving magnetic field. AMBR has been used to measure magnetic properties of magnetic particles, dynamic viscosity, detect bacterial cells with single cell sensitivity, and for designing a portable sensor. Asynchronous rotation of microparticles has also been studied in a variety of other systems.
Superparamagnetic beads, which may be micron-sized beads, are typically composed of an inert polymer sphere embedded with superparamagnetic nanoparticles, and may have several advantageous properties for use as labels. The magnetic material of the superparamagnetic beads may be stable over time, and the beads themselves are typically stable over long term storage and under most physiological conditions. Biological samples typically have little, if any, naturally occurring magnetic material, thus reducing the likelihood of background interference (with rare exceptions, such as magnetotactic bacteria). Super-paramagnetic beads may therefore be readily manipulated by external magnetic fields, and can be quantitatively detected by a variety of methods.
Sandwich immunoassays are common assay techniques used to detect biological molecules. A sandwich assay includes three components: a solid phase to isolate the analyte from the solution; the analyte itself; and a label or indicator, which binds specifically to the analyte. This results in the analyte being “sandwiched” between the solid phase and the label. Some of the more frequently used labels include fluorescent molecules and enzymes.